Legionnaires' disease is a common name for one of the several illnesses caused by Legionella or Legionnaires' disease bacteria (LDB). Legionellosis is the condition of being infected by Legionella bacteria which can cause serious pneumonia. By far, most legionellosis is the result of exposure to contaminated building water systems. Each year, hundreds of thousands of people suffer from these infections and many tens of thousands die from legionellosis or its complications.
About forty eight Legionella species with 70 serogroups have been classified. L. pneumophila is responsible for about 80%-85% of Legionella infections and serogroups 1 and 6 are responsible for two-thirds of Legionella infections. Other isolates and serogroups also contribute to Legionella infections. There are 15 serogroups of L. pneumophila and about 70 serogroups in total for Legionella. Some of the Legionella isolates and serogroups that cause infection include L. longbeachae, L. bozemanii, L. micdadei, L. dumoffii, L. feeleii, L. wadsworthii, and L. anisa. Two other genera have been proposed: Fluoribacter blue-white fluorescing species such as L. bozemanii and Tatlockia for the species L. micdadei. 
Legionella is widely present at low levels in the environment: in lakes, streams, and ponds. Water heaters, potable water distribution systems, decorative fountains, spa baths, swimming pools, humidifiers, evaporative cooling water towers, and warm, stagnant water provide ideal conditions for the growth and transmission of the biological hazard. Warm, stagnant water provides ideal conditions for growth. At about 30° C.-50° C. (75°-122° F.) the microorganism can multiply significantly and rapidly within its protozoan host, mostly the aquatic protozoa including different genera of amoeba. Rust (iron), scale, and the presence of other microorganisms can also promote conditions that result in rapid growth of Legionella. 
Preventive measures include regular maintaining and cleaning of building water systems such as cooling towers and evaporative condensers to prevent growth of Legionella, which should typically include for example, twice-yearly cleaning and periodic use of chlorine or other effective disinfectants; maintaining domestic water heaters at 60° C. (140° F.); and avoidance of conditions that allow water to stagnate, as, for example, large water-storage tanks exposed to heat from sunlight that produce warm conditions favorable to high levels of Legionella and its protozoan host.
Detection of Legionella by a “Standard Method”, as mandated by many government-sponsored guidelines, codes of practice, standards, regulations or laws such as for example, the Occupational Safety and Health Administration (OSHA) guidelines, takes about 10 days, due to the long incubation time required to grow detectable Legionella. Thus, definitive confirmation of viable Legionella takes about ten days when using the Standard Method for detection. During this period, Legionella would have multiplied and spread in situ and at many instances the facilities may have to be shut down, resulting in production delays or limited occupation or evacuation and therefore, substantial economic losses. If detected earlier, and corrective measures taken earlier, economic loss may be minimized. According to OSHA specifications, a site may be considered potentially dangerously contaminated with Legionella bacteria if at least 10 colony forming units (CFU)/ml of Legionella are present in a drinking water distribution system or 100 CFU/ml in a cooling water system. In humidifiers, even 1 CFU/ml is considered potentially dangerous according to these OSHA guidelines.
For the Standard Method, buffered charcoal yeast extract (BCYE) medium is used to grow and culture Legionella. Several refinements and improvements resulted in the currently preferred BCYE medium that is enriched with α-ketoglutarate (Edelstein BCYE-α medium) with or without selective antimicrobial agents and indicator dyes. This medium can be supplemented with bovine serum albumin. For example, Legionella MWY selective supplement media from Oxoid Limited (United Kingdom; product code SR0118) includes per 100 ml of the medium, glycine 0.3 g; polymyxin B 5,000 IU; anisomycin 8.0 mg; vancomycin 100 μg; bromothymol blue 1.0 mg; and bromocresol purple 1.0 mg.
The Standard Method, as disclosed in the 1998 publication entitled “Water Quality Detection and Enumeration of Legionella”, by the International Organization for Standardization of Geneva, Switzerland, which is commonly referred to as the ISO 11731 standard, specifies use of the BCYE-α medium supplemented with ammonia-free glycine, vancomycin, polymyxin B, and cycloheximide (GVPC). In addition to these supplements, GVPC or BCYE contains ferric pyrophosphate, L-cysteine, and as indicated, α-ketoglutarate. This method is generally consistent with the original method developed by the Centers for Disease Control and Prevention and with standard methods used in Australia and Singapore.
A method that is substantially similar to these is used in France (AFNOR T90431). In Standard Methods, selectivity steps such as acid treatment and/or heat treatment are required to inhibit competition from faster growing bacteria that may overwhelm Legionella in the sample.
The Standard Method requires a protocol for obtaining the samples, shipping them back to an analytical laboratory, and utilizes a specialized medium. In the laboratory, the method requires spreading a small volume of sample (0.1 ml) onto the surface of buffered charcoal yeast extract agar supplemented with growth factors and antibiotics and then incubating the media and the sample at a constant temperature and humidity for up to 10 days. The long incubation time is necessary because Legionella bacteria grow slowly on this growth medium. Growth on the agar surface must be sufficient for a microbiologist to count the number of colony forming units (CFU) on the surface of the agar after about ten days of incubation. The CFU count is used to determine a viable cell concentration by computing the value per unit volume. For example, a plate with 10 CFUs from 0.1 ml of undiluted sample indicates a viable Legionella concentration of 100 CFU/ml sample.
Several factors, however, limit the use of the Standard Method culture. First, an analyst's experience with the Standard Method directly correlates with pathogen quantification. Second, the Standard Method requires ten days to yield confirmed results, owing to the slow growth of Legionella on agar plates and the required confirmation tests. Third, the preparation of the medium is error-prone and requires extensive quality control. Fourth, the pathogen is sensitive to factors that are difficult to control during sample transit. Fifth, the concentration steps used to achieve lower detection limits are inefficient and not always reliable e.g., less than 50% of viable Legionella is recovered during sample concentration processing. Sixth, the method requires growing the pathogen to an extent that produces many visible colonies each containing millions or billions of potentially infective disease-causing bacteria on the surface of the agar plates. This operation is dangerous and must be therefore performed by specially trained analysts in properly equipped laboratories to ensure the safety of the analysts and the surrounding community.
Other methods that are used, in addition to the above-described Standard Method, are molecular methods. Molecular methods are faster, less expensive, less subjective, more sensitive, and are capable of being performed in the field. However, they all suffer two critical limitations—none ofthe molecular methods, commercially available or otherwise, are able to 1) differentiate between viable, i.e., Legionella cells that can grow and be quantified under the conditions (media, incubation temperature) specified in the Standard Method, and the background of non-viable, or dead Legionella and 2) no quantitative determination of Legionella cells per unit volume (such as milliliters or liters) can be rendered from the data. Thus, in practice, only the above-mentioned Standard Method is able to detect the effect of disinfection of a contaminated or suspected site, because it is the only method that is capable of distinguishing between viable and non-viable Legionella bacteria and quantifying the hazard. Such differential and quantifiable detection is an essential requirement to confirm effective hazard control in engineered water systems. However, quantitative differentiation of viable Legionella is not a requirement in most clinical applications.
Molecular methods of Legionella detection include nucleic acid detection using the polymerase chain reaction (PCR) or fluorescence in situ hybridization (FISH), and serologic methods by antigen/antibody reactions detected with enzyme linked immuno-specific assays (ELISA) or differential fluorescent antibody direct cell counting. These molecular detection systems are useful in the clinical laboratory for diagnosis and sero-grouping Legionella. However, for environmental or industrial samples, nucleic acid or serological methods should be used only as a rapid screen to identify those samples that are completely free of any Legionella and not as a basis to detect or quantify viable and culturable Legionella. 
Some of the distinguishing attributes of the Standard Method are: 1) differentiating viable from non-viable Legionella; 2) measuring all culturable species and serogroups of Legionella; 3) providing a viable Legionella count that can be expressed per unit volume or weight of sample; 4) global recognition of validity.
Some of the severe limitations of the Standard Method are: 1) a long incubation period of ten days is required before CFUs can be visually counted because Legionella grow slowly on solid media; 2) storing agar plates for ten days during incubation requires significant incubator space and humidity controlled conditions; 3) the systems, such as cooling water, domestic water, soils, and the like from which samples have been taken, usually change very significantly during the ten day incubation period; 4) the act of growing biological hazards taken from the environment into visible colonies comprised of millions or billions more infective viable bacteria is dangerous and must be performed therefore, in a laboratory with trained persons and special equipment.; and 5) shutting down production in a facility contaminated or suspected to be contaminated with Legionella, closing the facility or restricting access to it for 10 days while waiting for confirmation that the biological hazard has been controlled results in significant economic loss. There are many examples of highly significant economic losses from such facility closures or restrictions.
A rapid detection system for Legionella that can quantify viable Legionella in viability units that are equivalent to those used in the Standard Method and is also capable of being used safely in a field setting is therefore desirable.